Abstract:[Background] Alginate lyase has various types and degradation mechanisms, and has become a tool for the efficient and environmentally friendly production of alginate oligosaccharides, as well as a research hotspot for high-value development and utilization of algae. [Objective] This study aimed to isolate alginate lyase-producing strains, determine the optimal fermentation conditions for enzyme production, and analyze degradation products to reveal the degradation characteristics of the enzyme. [Methods] The strain was screened from the sea mud near kelp farms using alginate as the sole carbon source, and was identified according to morphological observation, physiological and biochemical tests, and 16S rRNA sequence alignment. The fermentation medium was optimized and used for enzyme production. Products at different degradation degrees were analyzed by IR and anion exchange chromatography to explore the degradation preference of the enzyme, and the final products were characterized by TLC and LC-MS. [Results] An alginate lyase-producing Vibrio natriegens SK42.001 was screened and identified. The optimized fermentation medium contained 8.0 g/L alginate, 8.0 g/L (NH₄)₂SO₄, 30.0 g/L NaCl, 5.0 mmol/L MgSO₄·7H₂O and 10.0 mmol/L K₂HPO₄·2H₂O, and the fermentation activity was (5.20±0.14) U/mL. The alginate lyase produced by strain SK42.001 had a preference to degrade guluronic acid blocks, and the final products present a relatively simple degree of polymerization, which were predominantly trisaccharides. [Conclusion] A V. natriegens SK42.001 with high yield of alginate lyase was screened. The enzyme produced by the strain has a preference to degrade guluronic acid blocks and specifically produces trisaccharides, which has the potential to be further developed for large-scale production of alginate lyase and alginate oligosaccharide biological products.

Abstract:[Background] Microbes in deep-sea hydrothermal vents use the extensive H₂S and sulfides in the environment to form a close symbiotic relationship with macrofauna. For example, sulfur-oxidizing bacteria use their unique metabolic system to help their hosts better adapt to the extreme conditions in hydrothermal vents. However, there is still no study on the identification and functional analysis of these symbionts of vent fauna. [Objective] To investigate the diversity and sulfur oxidation characteristics of culturable sulfur-oxidizing bacteria in hydrothermal benthos, and explore the symbiosis between sulfur-oxidizing bacteria and their animal hosts. [Methods] We used three culture media (M1, SPG, SOB) to isolate sulfur-oxidizing bacteria associated with hydrothermal benthos from different areas in the deep-sea hydrothermal vents. 16S rRNA gene sequences were used for phylogenetic analysis. The iodometric method was used to measure the sulfur oxidation activity. [Results] In the experiment, we obtained 169 strains of sulfur-oxidizing bacteria, which belong to Actinobacteria and Proteobacteria. Among them, the genus Halomonas was dominant, accounting for 73%. Three isolates showed higher sulfur oxidation activities:Thioclava nitratireducens M1-LQ-LJL-11 (70.4%), Qipengyuania vulgaris M1-TC-YB-4 (77.7%), and Dietzia cercidiphylli M1-TC-01-YL-6 (69.8%). [Conclusion] The diversity of culturable sulfur-oxidizing bacteria in hydrothermal benthos was reported for the first time. It was found that Gammaproteobacteria was the dominant, which had high sulfur oxidation ability and could detoxificate H₂S for their hosts in the hydrothermal fluids.

Abstract:[Background] Picea crassifolia, an endemic plant with great ecological value in China, is an important tree species for forest regeneration and afforestation in desert mountain areas in Northwest China. The diverse symbiotic fungi in the roots of P. crassifolia contribute to its growth, development, and stress resistance. [Objective] The culturable symbiotic fungi were isolated from the roots of P. crassifolia on Helan Mountain and identified to reveal the fungal community composition. The results provide basic data for seedling breeding, afforestation, and ecosystem restoration of P. crassifolia. [Methods] The culturable symbiotic fungi in the roots of P. crassifolia were isolated directly from root tips and identified based on the morphological characteristics of colony and the rDNA ITS sequence. [Results] A total of 65 fungal strains were isolated from the roots of P. crassifolia, belonging to 18 species, 16 genera, 13 families, 8 orders, 4 classes of 1 phylum (Ascomycota). Phialocephala was the genus with the highest isolation frequency, with the isolates accounting for 31% of the total isolates. Phialocephala lagerbergi was the species with the highest isolation frequency (22%), followed by Cadophora interclivum (18%), Pleotrichocladium opacum (17%), Phialocephala fortinii (9%) and Alfoldi sp. (9%). The species and number of the symbiotic fungi varied at different development stages of P. crassifolia. The fungal isolation frequency in the roots of saplings, seedlings, and adult trees was 52%, 28%, and 20%, respectively. [Conclusion] The symbiotic fungi in the roots of P. crassifolia on Helan Mountain in Ningxia were diverse, the species and number of which varied at different development stages of the tree. All the isolated fungi in this study were obtained from P. crassifolia for the first time. The results are an important supplement to previous related studies, laying a foundation for further investigating the resources of culturable symbiotic fungi in the roots of P. crassifolia and exploring the role of symbiotic fungi in the process of P. crassifolia adapting to the alpine and arid environment.

Abstract:[Background] Phenol wastewater has attracted much attention as a kind of wastewater with strong toxicity and refractory degradation. At present, microbial fuel cell (MFC) have been widely used for the degradation of phenol wastewater. The power generation efficiency of MFC and the degradation efficiency of phenol are closely related to the microbial community in the reactor. [Objective] In order to improve the electricity generation effect of MFC and the degradability of harmful substances, it is necessary to explore the degradation of phenol in MFC and the structure of microbial community. [Methods] In open and closed circuit condition, high-performance liquid chromatography (HPLC) was used to test the removal rate of phenol by MFC; 16S rRNA gene high-throughput sequencing technology was used to analyze changes in the microbial community in the cathode compartment. [Results] The results showed that compared with open circuit control, the removal effect of phenol in closed circuit condition has been significantly improved. In addition, the species diversity of cathodic microorganisms has increased. Although the overall species abundance has decreased, the abundance of some electricity-producing and degrading bacteria has increased significantly, such as, Proteobacteria and Actinobacteria at the phylum level and Gammaproteobacteria at the class level. [Conclusion] The existence of the micro electric field promotes the growth of some functional microorganisms, which has a positive impact on the degradation of phenol and the power generation of MFC.

Abstract:[Background] Armillaria are medicinal and edible fungi having a parasitic or saprophytic phase, and it is vital to study the differentially expressed genes in these fungi. However, there are few reports on the internal reference genes of them. [Objective] To screen out the optimal internal reference genes for Armillaria mellea 541 under different experimental conditions. [Methods] In this study, ACT-1, α-TUB, β-TUB 1, γ-TUB, UBQ, EF-1γ, 18S rRNA BP, and GAPDH were used as candidate reference genes of A. mellea 541. The A. mellea 541 hypha (AH) and rhizomorph (AR) cultured on potato dextrose agar (PDA) medium were used as the control group, and those cultured on the PDA medium supplemented with diphenyleneiodonium chloride and sawdust as inhibitor group and inducer group, respectively. The expression levels of the eight genes were evaluated by RT-qPCR and BestKeeper. [Results] EF-1γ was selected as the optimal reference gene for A. mellea 541 because of its stable expression under different experimental conditions. [Conclusion] This study provides a reliable reference gene for analyzing the gene expression in Armillaria spp..

Abstract:[Background] Norovirus, featuring genetic diversity, is a major foodborne initiator of acute gastroenteritis in human worldwide. GⅡ.17, the most destructive genotype, is a novel variant emerging in Asia from 2014 through 2015 and has been predominant in Asia. Virus-like particles (VLPs) of Norovirus, prepared by genetic engineering, are safe with high immunogenicity, which are promising candidate vaccine of Norovirus. [Objective] This paper aims to prepare GⅡ.17 VLPs in Escherichia coli. [Methods] The gene encoding GⅡ.17 capsid protein VP1 was synthesized and cloned into pET28a-MsyB vector. The recombinant plasmid was transformed into E. coli BL21(DE3) and expressed under the induction of IPTG. The fusion tags were removed by tobacco etch virus (TEV) protease, followed by purification with His-Tag nickel column to yield the natural VP1 protein. SDS-PAGE, Western blot, and transmission electron microscope were employed to determine the reactogenicity and structure of VP1 protein. [Results] MsyB-VP1 can be expressed in soluble form, and the optimum conditions are as follows:IPTG at 0.8 mmol/L, and 37℃ for 8 h. The purified VP1 protein digested by TEV protease can specifically bind to mouse anti-GⅡ.17 VP1 polyclonal antibody and assemble into VLPs (diameter:about 40 nm). [Conclusion] In this study, GⅡ.17 VLPs were prepared with E. coli, which can be used as the candidate GⅡ.17 vaccine.

Abstract:[Background] Sialidases are a type of glycoside hydrolases that hydrolyze the terminal sialic acid residue from sialic acid-containing complex. Sialidases are ubiquitous in animals and microorganisms and have important biological functions.[Objective] To clone a novel sialidase gene blsia42 from Bifidobacterium longum express it in Escherichia coli BL21(DE3), and characterize the enzymatic properties of the expressed protein. [Methods] A novel sialidase gene blsia42 was cloned from B. longum, and the recombinant expression plasmid pET-28a-blsia42 was constructed and expressed heterologously in E. coli BL21(DE3). After the crude enzyme was purified by Ni-NTA affinity chromatography, the enzymatic properties were studied. [Results] The specific activity of purified BlSia42 was determined to be 164 935.2 U/mg. The molecular weight of BlSia42 was determined as 42.8 kDa and 41.5 kDa by SDS-PAGE and gel filtration, respectively. The optimum conditions for BlSia42 were pH 6.0 and 50℃, and this enzyme was stable within pH 3.5-9.0 and below 45℃. BlSia42 showed a broad range of substrate specificity and had hydrolysis activity towards α2,3, α2,6, and α2,8 glycosidic bonds. The activity of BlSia42 with 3'-SL and colominic acid as substrates was 87.50% and 67.19% of that with 6'-SL as the substrate. After colominic acid was hydrolyzed by BlSia42 for 12 h, the sialic acid concentration and hydrolysis rate was 2.4 g/L and 23.6%, respectively. [Conclusion] The excellent enzymatic properties make BlSia42 potentially suitable for the preparation of sialic acid and its derivatives.

Abstract:[Background] Alkaline phosphatase (ALP), a tool enzyme, is widely used in various fields. Among the ALPs, PhoA has been extensively applied in immunological detection, while the functions of PhoD in immunological detection have been rarely reported. [Objective] This paper aims to screen a bacterial strain producing high-activity PhoD, clone and express the enzyme gene phoD, and study the enzymatic properties of PhoD, which is expected to lay a foundation for the application of PhoD in immunological detection. [Methods] Soil samples rich in organic matter were used to isolate bacteria in organophosphorus plate, and 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) was employed to detect the enzyme activity of single colonies. The strain with high enzyme activity was selected and the phoD gene was cloned. [Results] S2-4, the strain producing high-activity ALP, was screened out, which was identified as Bacillus amyloliquefaciens by 16S rRNA gene alignment. Its phoD gene was cloned and induced, and the enzymatic properties of purified PhoD are as follows:optimal reaction temperature, pH, and Ca²⁺ concentration of 70℃, 9.8, and 3 mmol/L, respectively, inhibition of Mg²⁺ on PhoD activity, no significant influence of K⁺, Zn²⁺, Mn²⁺, and Fe²⁺ on PhoD activity, Michaelis constant (K_m), maximum reaction rate (V_max), and catalytic constant (k_cat) of 5.94 mmol/L, 31.46 μmol/(L·min), and 103.59 s^-1 at 25℃ in the presence of p-NPP, separately. The k_cat was 1.60 folds that of Escherichia coli alkaline phosphatase (EAP). [Conclusion] S2-4 can synthesize alkaline phosphatase with high activity and its monomer enzyme PhoD has higher catalytic efficiency than EAP, which is more advantageous than EAP as a marker enzyme.

Abstract:[Background] Morel (Morchella spp.), a worldwide species, has important scientific and economic value. However, there are few studies on the correlation among elements of its rhizosphere microecosystem. [Objective] We explored bacterial community-soil physico-chemical property and bacterial community-enzyme activity relationships in rhizosphere soil of wild morels in different areas of Gansu Province. [Methods] With Illumina MiSeq technology, the bacterial community structure in the rhizosphere soil was measured and then bacterial diversity was analyzed. On this basis, the above relationships were elucidated.[Results] The pH of rhizosphere soil decreased with the rise of elevation, but the major and minor elements and enzyme activity varied irregularly with sampling site. Proteobacteria and Actinobacteria dominated at all the sampling sites. The bacterial operational taxonomic units (OTUs) and Shannon's diversity index at LX were more higher than those at other sites. Redundancy analysis (RDA) showed that Proteobacteria, Bacteroidetes, and Verrucomicrobia were positively correlated with soil nutrients, soil water content, and elevation, but negatively correlated with soil pH and content of Ca and Mg. Actinobacteria, Acidobacteria, and Planctomycetes were in positive correlation with Mg content, soil urease, and arylsulfatase, but in negative correlation with the content of Fe, Se, and available potassium, soil dehydrogenase, and alkaline phosphatase. Gemmatimonadetes and Chloroflexi were positively correlated with soil pH and Ca content, whereas Firmicutes showed positive correlation with the content of Fe and Se. [Conclusion] We clarified the correlation among elements in the rhizosphere soil microecosystem of wild morels in Gansu, laying a theoretical foundation for efficient cultivation of morel.

Abstract:[Background] Phosphorus is a macronutrient for plant growth, but most of it cannot be absorbed by plants. Phosphate-solubilizing microorganism (PSM) can secrete organic acids to dissolve the insoluble soil phosphates, improve plant growth, crop yield and quality by increasing phosphorus utilization.[Objective] The research aims to study the physiological functions of pqqE and GDH genes in the Pseudomonas fluorescens CLW17 strain. [Methods] Bioinformatics analyses have been done for two gene encoding proteins using bioinformatics software. We obtain two deletion mutant strains (CLW17ΔpqqE and CLW17ΔGDH) of pqqE and GDH gene by homologous recombination technology, and the corresponding supplementary strains (ΔpqqE/pqqE and ΔGDH/GDH) were obtained using the combined transfer method. The phosphate solubilizing abilities and organic acid production abilities of wild-type, mutant strains, and complementary strains were detected by NBRIP medium, molybdenum anti colorimetry method, and high-pressure liquid chromatography (HPLC) method, respectively. [Results] The numbers of amino acids encoded by pqqE and GDH genes were 390 and 803, respectively. There is no signal peptide for both genes. pqqE had no transmembrane domain, while GDH predicted five transmembrane domains. The pqqE and GDH genes are the phosphate-solubilizing genes of the CLW17 strain, and the deletion of the two genes greatly reduces the phosphate-solubilizing ability of this strain, moreover, the complementary strains of these two genes can restore the phosphate-solubilizing ability. Wild strain CLW17 can secrete a variety of organic acids, among which gluconic acid content is the highest, followed by acetic acid. The ability to produce organic acids in the knocked-out strain was greatly reduced, especially the ability to produce gluconic acid and acetic acid. [Conclusion] The pqqE and GDH genes are the key genes for the phosphate solubilization of the CLW17 strain, the phosphate solubilization ability of the strain is closely related to the production of organic acids, especially gluconic acid and acetic acid. This study laid the foundation for further study on the interaction and phosphorus solubilization mechanism between the strain and Taxus chinensis var. mairei.

Abstract:[Background] L-amino acid supports the growth and development of Lentinula edodes as a nutrient. It is of great significance for the high-production and high-quality development of L. edodes industry to optimize the growth substrate of L. edodes by adding amino acids. [Objective] Exogenous alanine (Ala), serine (Ser), and asparagine (Asn) were used to improve the medium, and the possible metabolic pathway of the compound amino acids to promote the growth of L. edodes mycelia was explored. [Methods] The three-factor, three-level Box-Behnken design was employed to optimize the concentration of the amino acids, with the evaluation indicator of mycelial growth rate, and a series of physiological indexes of L. edodes mycelia in the optimized medium were determined. [Results] The theoretical optimal concentration of Ala, Asn, and Ser was 174.711 mg/L, 113.826 mg/L, and 5.661 mg/L, respectively, and the formula was verified to effectively promote the growth of L. edodes mycelia. Compared with potato dextrosa agar (PDA), the optimized medium enabled high content of pyruvic acid (PA) and protein, significantly high activity of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), and low content of malondialdehyde (MDA) in L. edodes mycelia. [Conclusion] The study provides a theoretical reference for the improvement of L. edodes medium and the mechanism of amino acids promoting mycelial growth.

Abstract:[Background] The molecular mechanism of fruiting body development in mushroom-forming fungi, especially the transition from mycelium to primordium, is still unclear. Most of related studies focused on limited fungal species or environmental factors, and offered little information on the key genes in the development of mushroom-forming fungi. [Objective] This paper aims to study the molecular mechanism for primordium formation of mushroom-forming fungi. [Methods] RNA-seq data of 11 fungal species and 4 related environmental factors were analyzed. [Results] The number of up-regulated genes ranged from 325 (Pleurotus tuoliensis) to 2 854 (Rickenella mellea), while that of down-regulated genes was in the range of 379 (Pleurotus tuoliensis) to 3 189 (Armillaria ostoyae) compared with that of the control. As for the gene ontology (GO) terms, the top three biological processes were oxidation-reduction process, metabolic process and carbohydrate metabolic process. The mainly involved cellular components were the integral component of membrane, nucleus, and membrane, and the related molecular functions were hydrolase activity, oxidoreductase activity and catalytic activity. [Conclusion] There are some common key pathways for the primordium formation among mushroom-forming fungi.

Abstract:[Background] The drought-resistant sand-fixing shrub Artemisia ordosica, which is widely distributed in the desert area in northern China, plays an important role in stabilizing the ecosystem in desert area. [Objective] Endophyte plays an essential part in plant life. It is of great significance for understanding the interaction among microbial communities and protecting host plants against biotic and abiotic stresses to clarify the structure variation of endophytic communities in young and mature tissues of plants during growth and development. [Methods] In this study, we measured the lignification indices of young and mature branches of A. ordosica from Labahu Forest Farm in Ningxia and performed high-throughput sequencing of the endophyte communities. [Results] Content of lignin and cellulose was significantly different between A. ordosica branches at different development stages. The diversity of endophytic bacteria and fungi in young branches was higher than that in mature branches, and more OTUs were found in the former than in the later. Mature branches accumulated lignin and cellulose, thus boasting stronger adaptability. With the development of branches, endophytic fungal community changed more significantly, as the unidentified phylum gradually took the position of Ascomycota as the dominant phylum. [Conclusion] Development stage of A. ordosica branches has profound impact on the assembly of plant microbiome. Endophytic bacteria and fungi play different ecological roles at different development stages of plants. We revealed the difference in the community structure of endophytic bacteria and fungi between young and mature branches of A. ordosica, hoping to provide a reference for ecological restoration in desert area.

Abstract:[Background] Plant growth-promoting agents have been widely used in agricultural production, while the knowledge to their impacts on the root microbial community of crops in substrate culture is limited. [Objective] The objective is to investigate the effects of Pseudomonas sp. JP2-3 and Bacillus subtilis Z54 on the growth and root bacterial community of tomato in substrate culture.[Methods] The two strains were inoculated by root dipping and root irrigation for the tomato plants in substrate culture. We then measured the growth indexes of tomato seedlings to explore the effect of growth promoting bacteria on tomato growth. The strain effects on the composition and structure of the root bacterial community were analyzed by 16S rRNA gene amplicon sequencing technology.[Results] The two strains promoted tomato growth. Four growth indexes (plant height, stem diameter, maximum leaf length, and maximum leaf width) in strain JP2-3 group were higher than those in CK group. Except stem diameter, the other three indexes in strain Z54 group were higher than those in CK group. Both strains JP2-3 and Z54 changed the α diversity of bacterial community and decreased the bacterial richness and diversity compared with the CK group. Particularly, the decrease was more significant in strain Z54 group (P<0.05). The β diversity of root bacterial community was significantly changed by strain Z54 while slightly affected by strain JP2-3. The dominant bacterial phyla included Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes, and the dominant genera were Streptomyces, Pseudomonas, Ideonella, Devosia, etc. The composition of root bacterial community of tomato in substrate culture was similar to that in soil culture at the phylum level while different at the genus level. Inoculation with strain JP2-3 increased the relative abundance of Ideonella, Actinoplanes, and Aquincola, while strain Z54 increased the relative abundance of Brevundimonas and Flavobacterium. Among the genera with increased relative abundance, many bacteria are beneficial to plants. LEfSe showed that the biomarkers of strain JP2-3 group were mainly distributed in Betaproteobacteria and Actinobacteria, while those of strain Z54 group in Gammaproteobacteria. [Conclusion] Pseudomonas sp. JP2-3 and B. subtilis Z54 promoted the growth of tomato in substrate culture and had different impacts on the composition and structure of the bacterial community in tomato roots. The two strains increased the relative abundance of indigenous beneficial microorganisms and cooperated with indigenous beneficial microorganisms to promote tomato growth.

Abstract:[Background] The increasingly serious root rot has posed a threat to the production of naked barley (Hordeum vulgare L. var. nudum Hook. f.) and hampered the prevention and control of the disease and the development of the naked barley industry in Qinghai province. However, the root rot of naked barley has been rarely studied and the pathogens are unclear. [Objective] This paper aims to clarify incidence and pathogens of the root rot in naked barley and the pathogencity of the pathogens, which is expected to lay a theoretical basis for the prevention and control of the disease. [Methods] The conventional method of tissue isolation was adopted to isolate pathogens of root rot in naked barley. The pathogens were detected by both morphological identification and molecular identification, and their pathogenicity was determined with a beaker of water-agar. [Results] A total of 4 stains with strong and significantly different pathogenicity were isolated, which were identified as Clonostachys rosea. As verified by Koch's postulates, they were new pathogens of root rot of naked barley, and the induced root rot was found for the first time at home and abroad.[Conclusion] C. rosea can cause root rot in naked barley with strong pathogenicity.

Abstract:[Background] Tomato bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum, which severely restricts the production of tomato. [Objective] To screen out an antagonistic bacteriumand apply it to biocontrol of tomato bacterial wilt. [Methods] We used the inhibition zone method and agar diffusion method to screen the bacterial strains with strong antagonistic effect against R. solanacearum from the rhizosphere soil of healthy tomato plants in a field with bacterial wilt in Hengyang, Hunan. The strain was identified by the observation of morphological characteristics, physiological and biochemical tests, and sequence analysis of 16S rRNA gene and gyrA gene. The fermentation conditions were optimized by single factor test and orthogonal test. The control effect was investigated by field experiment. [Results] Strain TR-1 with a significant antagonistic effect against R. solanacearum was screened out and preliminarily identified as Bacillus velezensis. The optimized medium for TR-1 was composed of 20.0 g/L soluble starch, 10.0 g/L soybean peptone, and 5.0 g/L K₂HPO₄. The optimum fermentation conditions:pH 6.0-7.0, 30-33℃, 160 r/min, and 48 h. The inhibition zone of the sterile fermentation supernatant of TR-1 cultured under the optimized conditions against R. solanacearum showed a diameter of 2.95 cm, which was about twice the size before. TR-1 showed the control effect of 60.30% in the field experiment. [Conclusion] The optimization of fermentation conditions greatly improves the inhibition effect of TR-1 fermentation broth. The strain has a good control effect on tomato bacterial wilt in the field experiment and demonstrates the potential to be applied in the biocontrol of this disease.

Abstract:[Background] Gut microbiota is closely related to the health and environmental adaptability of the host. Yak (Bos grunniens) is a herbivorous ruminant unique to the Qinghai-Tibet Plateau. The effect of altitude on the gut microbiota structure of yaks and the roles of gut microbiota in the adaption of yaks to high altitude, however, are still largely elusive. [Objective] This paper aims to explore the diversity of gut microbiota of yaks grazing on the Qinghai-Tibet Plateau and the relationship with altitude. [Methods] A total of 22 fresh fecal samples were collected from yaks in Maqin (altitude:4 220 m) and Ledu (altitude:2 745 m) of Qinghai, and the V3-V4 region of 16S rRNA gene was amplified with universal primers and then subjected to high-throughput sequencing and analysis. [Results] A total of 1 723 018 valid reads were obtained from the 22 samples, which were clustered into 1 113 operational taxonomic units (OTUs) based on 97% similarity. The OTUs belonged to 263 genera and 22 phyla. Firmicutes, Bacteroidetes, Ruminococcaceae_UCG-005, and Rikenellaceae_RC9_gut_group dominated the samples from the two regions, despite the significant difference in abundance. As the altitude elevated, the abundance of Firmicutes and Ruminococcaceae_UCG-005 significantly increased, while that of Bacteroidetes and Rikenellaceae_RC9_gut_group significantly decreased. As for the structure and difference of gut microbiota, gut microbiota structure of yaks was dramatically affected by altitude. In addition, PICRUSt2 analysis revealed that related metabolic pathways in gut microbiota such as energy metabolism, cofactor and vitamin metabolism, nucleotide metabolism, and glycan and amino acid metabolism were significantly enriched in yaks grazing at the higher altitude, in part because yaks improved forage utilization efficiency and harvested more energy to adapt to the extreme environment at higher altitude.[Conclusion] The gut microbiota structure of yak grazing at different altitudes was obviously different, and gut microbiota was proposed to play an essential role in the adaption of yaks to the environment at high altitude.

Abstract:[Background] Bovine viral diarrhea virus (BVDV) is among the important pathogens of calf diarrhea. However, there are few studies on the interaction mechanism between BVDV and host factors, which impedes the prevention and control of BVDV. [Objective] This study aims to explore the effect of vesicle-associated membrane protein A (VAPA) on BVDV replication. [Methods] According to the information of VAPA gene in GenBank, we designed the small guide RNA (sgRNA) targeting VAPA using Benchling and CHOPCHOP, cloned it to lentiCRISPR v2 vector, and then packed the lentivirus. Afterward, Madin-Darby bovine kidney (MDBK) cells were transfected with the lentivirus, followed by puromycin screening for 5 generations and detection of VAPA knockout (KO) by Western Blot. Total RNA was extracted from VAPA KO cells at different time after BVDV infection and then reverse-transcribed into cDNA. Real-time quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) were used to detect the 5'-untranslated region (UTR) mRNA level and the accumulation of double strand RNA (dsRNA) of BVDV, respectively. Cytopathic effect (CPE) was observed at different time after BVDV infection. Progeny virus titer was calculated with the Reed-Muench method. The same number of VAPA KO and scramble control cells were inoculated and then the living cells were counted at different time after virus infection to detect whether VAPA KO affected the cell viability. [Results] After lentivirus infection and puromycin screening, the results of Western Blot showed that VAPA protein was significantly decreased and thus VAPA KO cells were successfully established. The 5'-UTR mRNA level and dsRNA accumulation in VAPA KO cells infected with BVDV were significantly reduced compared with those of the scramble control cells. The CPE caused by BVDV infection was significantly delayed and attenuated and the titer of progeny virus decreased significantly at 12 h and 36 h and highly significantly at 48 h. The viability of VAPA KO cells was not affected compared with that of the scramble control cells. [Conclusion] VAPA KO can significantly inhibit BVDV replication. Therefore, this study provides an important target for the prevention and control of BVDV.

Abstract:[Background] Establishing the diarrhea model is a common method to study the mechanism of bacterial diarrhea and anti-diarrhea mechanism. [Objective] Five strains of Escherichia coli with different serotypes were isolated from the feces of calves with diarrhea. The diarrhea model of mice was established by intragastric administration. [Methods] Kunming mice were randomly divided into six groups:normal control (NC) group, E. coli O1, E. coli O2, E. coli O8, E. coli O78, and E. coli O86 groups, with 12 mice in each group. The number of E. coli in all the challenge groups was 3×10¹³ CFU/mL, and the strain liquid was administrated at 0.2 ml/10 g body weight and twice/day for 7 continuous days. The body weight and diarrhea rate of mice were recorded on days 3, 5 and 7, and the serum samples of mice in each group were collected for the determination of interleukin 1β (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), immunoglobin G (IgG), IgA, and secretory immunoglobulin A (sIgA) levels. The duodenum of mice in each group was collected on the 7th day to prepare H&E sections, and the cecum contents were collected for 16S rRNA gene analysis. A diarrhea model was established with E. coli O₈ via intragastric or intraperitoneal administration, and ofloxacin was used to treat the mice with diarrhea. [Results] On days 5 and 7, the mice in the E. coli groups showed lower body weight (P<0.05) and higher diarrhea rate than those in the NC group. HE staining results showed that the intestinal villi of mice in E. coli groups showed different degrees of damage, and E. coli O2 and O8 caused the most serious damage. The serum levels of IL-1β, IL-6, and TNF-α significantly elevated after challenge (P<0.05), especially in the E. coli O1 and O8 groups. However, the levels of IgG, IgA, and sIgA in the challenge groups significantly decreased compared with those in the NC group (P<0.05), especially in the group challenged with strain O8. The 16S rRNA gene analysis of cecum contents in mice showed that compared with the NC group, the challenge groups had decreased alpha diversity of bacteria. Specifically, the abundance of Firmicutes decreased while that of Bacteroides, Proteobacteria and Actinobacteria increased in the challenge groups. All the mice died within 12 h after intraperitoneal injection with E. coli O₈, and antibiotic did not reverse this death, while the mice with diarrhea caused by intragastric administration could restore to normal after antibiotic treatment. [Conclusion] The five strains of E. coli from dairy calves with diarrhea could cause different degrees of diarrhea in mice after intragastric administration. Among them, E. coli O2, O8, and O1 caused serious damage to the intestinal villi of mice. The serum levels of IL-1β, IL-6 and TNF-α in mice were higher after administration with E. coli O8. Therefore, intragastric administration of 3×10¹³ CFU/mL E. coli O8 at 0.2 mL/10 g and twice/day for 7 continuous days could well establish the diarrhea model of mice, which can be used to evaluate drug efficacy.

Abstract:[Background] Salmonella typhimurium is a major zoonotic pathogen, and its multi-drug resistance is becoming increasingly serious. The two-component system can regulate the resistance of S. typhimurium. [Objective] We constructed the baeR-overexpressing strain and complementary strain of S. typhimurium to study the effect of BaeSR on the antibiotic resistance of S. typhimurium. [Methods] a complementary strain (CRcbaeR∆baeSR∆acrB) and an overexpression strain (CRpbaeR∆baeSR∆acrB) were constructed based on a baeSR and acrB double-deletion strain of S. typhimurium (CR∆baeSR∆acrB), and their antimicrobial susceptibility and biological characteristics were tested. RNA-Seq was used to screen the differentially expressed genes (DEGs) related to drug resistance, and RT-qPCR was conducted to verify some drug-related genes. [Results] The baeR-overexpressing strain and complementary strain were successfully constructed. Compared with that in CR∆baeSR∆acrB, the minimum inhibitory concentrations (MICs) of ofloxacin, enrofloxacin, florfenicol, mequindox, ceftazidime, ceftiofur, amoxicillin, and ampicillin increased by 2-256 folds while that of spectinomycin and apramycin decreased by 50% in CRpbaeR∆baeSR∆acrB. The MIC of ceftazidime, ceftiofur, florfenicol, and enrofloxacin was two-fold higher in CRcbaeR∆baeSR∆acrB than in CR∆baeSR∆acrB. The growth curves of CR∆baeSR∆acrB, CRpbaeR∆baeSR∆acrB, and CRcbaeR∆baeSR∆acrB showed no significant difference. However, the biofilm-forming ability and motility of CRpbaeR∆baeSR∆acrB and CRcbaeR∆baeSR∆acrB were significantly higher than those of CR∆baeSR∆acrB (P<0.01). A total of 743 DEGs were identified between CRpbaeR∆baeSR∆acrB and CR∆baeSR∆acrB, including 724 upregulated genes and 19 downregulated genes. Bioinformatics analysis showed that the DEGs were mainly enriched in pathways such as β-lactam resistance, quorum sensing, and two-component system. A total of 3 073 DEGs were identified between CRcbaeR∆baeSR∆acrB and CR∆baeSR∆acrB, including 1 467 upregulated genes and 1 606 downregulated genes. These DEGs were mainly enriched in pathways such as carbon metabolism, biosynthesis of amino acids, and biosynthesis of antibiotics. A total of 15 drug resistance-related DEGs were screened out, mainly involving efflux pump, biofilm, and two-component system. Five out of the 15 DEGs were randomly selected for RT-qPCR, which showed consistent results and confirmed the reliability of RNA-Seq results. [Conclusion] This study indicates that baeR affects the drug resistance of S. typhimurium by regulating its biofilm-forming ability and motility. RNA-Seq screened out 15 resistance-related DEGs, indicating that baeR overexpression can affect the drug resistance of S. typhimurium by regulating the expression of the genes associated with efflux pump, biofilm and flagella.

Abstract:[Background] Resistance genes can be transferred among environment, animal, and human, and transferred for a long distance by migratory birds, thus posing a great threat to public health. [Objective] We isolated and identified the main Enterobacteriaceae bacteria from the feces of migratory birds in Nansha Wetland Park, Guangzhou, and explored the resistance to common antibiotics and the main resistance extended-spectrum beta-lactamase (ESBL) genes. On this basis, we assessed the risk of migratory birds in Guangzhou carrying and transferring resistance bacteria and genes. [Methods] From January to December 2019, 393 fecal samples were collected, and the bacteria were isolated, cultured, and identified. Through drug susceptibility test, the resistance of the bacteria was detected. PCR was performed to amplify the resistance genes bla_CTX-M, bla_TEM, and bla_SHV, followed by sequencing and BLAST alignment. [Results] A total of 59 Enterobacteriaceae strains (isolation rate 15.01%) were detected from the 393 fecal samples, among which Pantoea agglomerans (36 strains, 61.02%) was most abundant. Of the 59 strains, 89.83% were resistant to the tested antibiotics, and the resistance rates to cefazolin (81.36%), sulfisoxazole (52.54%), and ampicillin (44.07%) were particularly high. Moreover, 55.93% of the strains were resistant to multiple antibiotics. In addition, 7 strains were resistant to carbapenems. The sequencing result showed that 23 strains carried the resistance gene bla_TEM-1 (5.85%). [Conclusion] Enterobacteriaceae bacteria in the feces of migratory birds in Nansha Wetland Park, Guangzhou are intricacy, and the rates of drug resistance and multi-drug resistance are high. The result suggests the risk of the migratory birds carrying carbapenem-resistant Enterobacteriaceae bacteria, which has important public health significance.

Abstract:[Background] Vibrio parahaemolyticus is an important foodborne pathogen worldwide, which can cause gastroenteritis. Quorum sensing system LuxS/AI-2 can regulate the biological characteristics of bacteria, which provides a new pathway to study the dissemination mechanism and control techniques of V. parahaemolytics. [Objective] To study the effect of quorum sensing signaling molecule autoinducer-2 (AI-2) on the biological characteristics of V. parahaemolyticus Vp2009027 from seafood. [Methods] The critical gene luxS related to the synthesis of AI-2 was knocked out, and the luxS-knockout mutant of V. parahaemolytics Vp2009027 was constructed by homologous recombination with suicide plasmid. The effect of LuxS/AI-2 on the biological characteristics of V. parahaemolyticus was determined by comparing the growth, AI-2 activity, motility, biofilm formation ability, and antibiotic resistance between the wildtype strain and the mutant. [Results] The mutant was constructed successfully, which showed no significant difference in growth compared with the wildtype strain. The deletion of luxS led to the failure of AI-2 synthesis, increase in motility and biofilm formation ability, and weakening of tetracycline resistance. [Conclusion] luxS gene could regulate the biological characteristics of V. parahaemolyticus, which provided a reference for further research on the dissemination mechanism and development of control techniques of V. parahaemolyticus.

Abstract:[Background] Superficial mycosis cases have been increasing year by year and the pathogenic fungi have become one of the major threats to human health. [Objective] This paper aims to explore the inhibitory effect of dictamnine (DIC) on Trichophyton mentagrophytes and the underlying mechanism. [Methods] Transcriptome sequencing was performed on T. mentagrophytes and T. mentagrophytes after the action of DIC by high-throughput sequencing technology, processing and bioinformatics analysis of the sequencing sequence, sequencing quality evaluation and sequence annotation. By comparing with the control group, differentially expressed genes were identified, and then GO function significance analysis and KEGG metabolic pathway analysis were performed. [Results] The minimum inhibitory concentration (MIC) of DIC on T. mentagrophytes was 50 μg/mL. A total of 360 differentially expressed genes (DEGs) were identified between the control group and DIC high-dose group (265 up-regulated and 95 down-regulated), and the expression of MFS1, KU70, KU80, L2, cpaT, MFS2, VdtG and patC was significantly different. The DEGs were mainly involved in the GO terms of ribosome, mitochondrial membrane, antioxidant activity, toxin metabolic process, single biological metabolic process, secondary metabolic process, primary active transmembrane transporter activity, and active transmembrane transporter activity. The changes in the KEGG metabolic pathway mainly focus on enrichment pathways such as ABC transport, membrane transport barriers, glutathione metabolism, DNA replication and repair changes, meiosis, oxidative phosphorylation and pyruvate metabolism disorders, and related genes are all There is a significant change (P<0.05). [Conclusion] DIC inhibits T. mentagrophytes by influencing DNA replication, expression, and repair, energy metabolism, ABC transport, and other pathways of the fungus.

Abstract:A variety of microorganisms constitute the normal microbiota in human reproductive tract. Lactobacillus is predominant among the microorganisms in reproductive tract of healthy people as it maintains the ecological balance of reproductive tract and prevents the invasion of pathogens. Disruption of the micro-ecological environment can lead to various diseases such as bacterial vaginosis, sexually transmitted infection, adverse pregnancy outcome, infertility and tumor. In addition to conventional antimicrobial therapy, probiotics play an important role in restoring community structure and maintaining reproductive health in response to reproductive tract inflammation. This article reviewed the research progress on the correlation between reproductive tract microecology and reproductive health in men and women.

Abstract:Influenza virus is one of the most important viruses that threaten human health for a long time. The inactivated influenza vaccine mainly produces strain-specific antibodies against the viral hemagglutinin. When the newly emerging influenza virus strain does not match the vaccine strain, the effectiveness of the vaccine will be greatly reduced. Due to the continuous emergence of antigenic drift and mutation of seasonal influenza viruses, people urgently need to find a wider range of protection methods. Previous studies show the importance of cellular immunity, especially the pre-existing memory T cells. Pre-existing memory T cells which target and recognize the conserved proteins inside influenza viruses can cross-react with influenza viruses of multiple subtypes, providing a certain degree of protection to influenza virus infections of the same subtypes or different subtypes. This article reviews the research progress of influenza virus cross-reactive memory T cells and some guiding applications of cross-reactive memory T cells in influenza prevention, aiming to provide a new reference for influenza prevention and vaccine development.

Abstract:Anaerobic ammonium oxidation (anammox) bacteria, which convert NH₄⁺ and NO₂^- or NO into NO₃^- and N₂ under anaerobic conditions, feature high efficiency and low energy consumption in the treatment of low carbon-nitrogen ratio wastewater, thus attracting great attention. At present, pure culture of anammox bacteria has not been realized, and there has been an explosion of research on anammox bacteria and the interaction with other bacteria in anammox community by meta-omics approaches. In this paper, we introduced the taxa and characteristics of anammox bacteria, reviewed the nitrogen metabolism and carbon metabolism of them, discussed the interaction of them with other bacteria in the anammox community in metabolism and information exchange which are important for the stability of the community, and finally summarized the future research directions. The result is expected to serve as a reference for in-depth study of the interaction between anammox bacteria and other bacteria in the community and establishment of stable and efficient nitrogen-removing systems with anammox bacteria.

Abstract:Since the 1850s, human fecal microbiota transplantation (FMT) has been used in clinical practice in western medicine. It has been evidenced that FMT can alleviate gastrointestinal disorders such as Clostridium difficile infection (CDI). Moreover, some studies have concluded that FMT is effective in relieving depression by modulating the microbiota-gut-brain axis (MGBA), which, however, needs to be further verified. Therefore, this paper described the relationship between MGBA and depression and summarized MGBA-related mechanisms of depression:abnormalities in the autonomic nervous system, hypothalamic-pituitary-adrenal (HPA) axis, enteric nervous system, and circulatory system, and the "leaky gut" hypothesis. Based on the above mechanisms, this review listed the animal experiments and clinical trials on the treatment of depression with FMT in recent years and dissected the current and future status of FMT, in an attempt to explore whether FMT is practical and reliable in the treatment of depression and provide ideas and methods for the treatment of depression. Despite the huge potential of FMT in the treatment of depression, more compelling research and experimental evidence are needed.

Abstract:As an important organ for digestion and absorption, intestinal tract is the largest immune organ of human body and plays a very important role in maintaining normal physiological activities. With the diversification of human diet, more attention has been attracted about the impact of intestinal flora on health. Intestinal microecology is related to more than 50 diseases including immunity, metabolic system diseases, tumors and so on. There is no doubt that diseases can also affect the human intestinal flora. Baizhu powder is a prescription mainly composed of edible medicinal plant materials, which can improve the intestinal microecology, invigorate spleen and replenish Qi, promote the secretion of saliva or body fluid and reinforcing the stomach, to achieve the purpose of maintaining human health. In recent years, many clinical studies and animal experiments have made remarkable progress in the treatment of intestinal flora disorders related diseases with Baizhu powder. This paper summarizes the mechanism of Baizhu powder on intestinal microecology, in order to provide some theoretical and experimental basis for clinical research on Baizhu powder in the treatment of intestinal related diseases.

Abstract:Gram-negative bacteria, such as the commonly used Escherichia coli strains, have poor protein secretion capacities due to their intricate double-layer membrane. This shortcoming limits the production of Gram-negative bacteria for specific biopharmaceuticals and other bioproducts. Therefore, the research on the protein secretion system of Gram-negative bacteria is of great significance. This review summarized the knowledge about protein secretion system of Gram-negative bacteria, including the secretion process, the types of secreted proteins, the structural information of the secretion system, and the technologies for protein secretion research. Thus, this review will provide referential information for studying the protein secretion mechanism in Gram-negative bacteria.

Abstract:A signal peptide is a short peptide fused in the recombinant protein N-terminus, generally composed of 15-30 amino acids, guiding the transport of recombinant protein to the periplasmic space of the cell. It is divided into N region, H region and C according to the structure and function. Recombined protein expression in Escherichia coli, often faces with the problem of forming an inclusion body. If the recombined protein is secreted into the periplasmic space, the periplasmic protein can not only help the recombination protein fold into to correct structure but also facilitate the production of disulfide bonds. This paper summarizes the structure composition, action structure and basic secretion pathways of signal peptides; discusses the efficient transport and screening methods of signal peptides. This paper also summarizes the new progress of recombination protein fusion signal peptides in E. coli to achieve perennial expression, and visions the screening techniques of signal peptides in the future.

Abstract:Straw is one of the most abundant biomass resources in the world. As the main utilization way of straw, straw incorporation is associated with the sustainable development of agriculture. Microorganisms are the most active bio-factors in the soil ecosystem, which are closely related to straw degradation, nutrient circulation, and soil health. To understand the micro-ecological effects of straw incorporation, we analyzed the soil enzyme activity, microbial biomass, culturable microbial populations, and diversity after straw incorporation. Furthermore, we compared the soil micro-ecological indicators under different conditions of straw incorporation, and discussed the limitations of the related studies. Finally, we put forward an outlook on the future research directions in this field. This study can provide a basis for safe straw incorporation.

Abstract:Oxalic acid, a metabolite with important functions, is ubiquitous in plants, animals, and microorganisms. Many fungi have been reported to be able to secrete oxalic acid, including plant pathogenic fungi, edible and medicinal fungi, and industrial fungi. Oxalic acid as a simple dicarboxylic acid is synthesized in fungi mainly through tricarboxylic acid cycle, glyoxylate cycle, and oxaloacetate pathway. Oxalic acid is an important biological factor produced by fungi, which not only affects the growth and development of fungi but also plays a role in fungus-plant interactions through toxin action, acidification of host plant tissue environment, participation in cell wall degradation, and induction of reactive oxygen species production. Therefore, it is essential in the life cycle and infection cycle of fungi. This paper reviews the properties and metabolism of oxalic acid, the oxalic acid-producing fungal species, the role of oxalic acid and oxalic acid-related genes in these species. Furthermore, we put forward the questions to be solved in the related research, aiming to provide scientific references for the utilization of the fungal resources producing oxalic acid and research ideas for the integrated management of plant fungal diseases.

Abstract:Curriculum ideology and politics is an important way of ideological and political education in universities in the new era. The history of science records the process from generation to sustainable development of scientific knowledge, which contains rich educational value and provides new perspectives and ideas for the ideological and political teaching of professional courses. In this study, we selected materials in four aspects of scientific spirit, scientific thinking, scientific interest, and scientific ethics from the rich educational values of history of science. Meanwhile, we reviewed the history of Nobel Prize related to Genetic Engineering. Then, taking the materials of the four aspects above as the carrier of education, we excavated the curriculum ideological and political elements contained in the history of Nobel Prize related to Genetic Engineering, and helps students achieve their goals in ideology and politics by carrying out the ideological and political education in the Genetic Engineering course. Finally, we employed questionnaire survey and in-depth interview to evaluate the teaching performance. With such teaching practice, we aim to guide students to establish the correct value concept and improve their ideological and political level, and to provide a reference for the construction of curriculum ideological and political system of biology courses.

Abstract:[Background] Aeromonas hydrophila is pathogenic to aquatic animals, livestock, poultry, and human. Hemolysin, aerolysin, and enterotoxin are major virulence factors particularly important in the early detection and prevention of A. hydrophila. There are few reports using colony PCR to extract templates for multiplex PCR. [Objective] Based on colony PCR, a multiplex PCR assay was established for rapid detection of five genes including hemolysin gene, enterotoxin gene, and 16s rRNA gene of A. hydrophila. [Methods] We used the selective Rimler-Shotts (RS) medium to enrich, isolate, and identify A. hydrophila in the sample. Subsequently, we established and optimized the multiplex PCR conditions for the 16S rRNA, ast, alt, aerA, and act of A. hydrophila and compared the results of multiplex PCR with the DNA templates extracted by different methods in colony PCR. Finally, we evaluated the specificity of the established method by using A. veronii, A. sobria and A. salmonicida. [Results] After 16S rRNA identification of single colonies on RS plates, the colony morphology of A. hydrophila and other cultivable bacteria was preliminarily characterized, and the enrichment degree of A. hydrophila was visually identified. The optimization results of the multiplex PCR system showed that the optimal primer concentration ratio was 16s rRNA:ast:alt:aerA:act=1:2:2:3:4. PCR for fresh bacterial liquids treated with both boiling-refrigerated centrifugation method and boiling-centrifugation method could produce clear bands, while the PCR of single colony required the former method. The established multiplex PCR method was specific. [Conclusion] Colony multiplex PCR can be used to easily and intuitively detect A. hydrophila and its virulence genes without the need of kit extraction of DNA templates, and it was specific.